Malignant hyperthermia (MH), a pharmacogenetic disorder of skeletal muscle elicited by exposure to volatile anesthetics and depolarizing muscle relaxants, is well recognized in humans and pigs, although a number of definite episodes have also been reported in dogs.1,2,3,4,5,6,7 When given these agents, MH susceptible (MHS) dogs exhibit tachycardia, hyperthermia, elevated carbon dioxide production, and death if the anesthetic is not discontinued. Specific interventions, including use of the calcium release channel antagonist dantrolene,8,9 are efficacious in reversing signs of the canine syndrome. In most reports of MH in dogs metabolic acidosis is moderate and muscle rigidity is minimal, in contrast to the severity of both in the swine or human condition. Many additional accounts of episodes resembling MH in dogs within the peri-operative interval,10,11,12,13, during exertion,14,15,16,17 or other chemical exposures18 have been published. Monitoring and laboratory investigations in these descriptions are scant, and uncertainties persist regarding the incidence of canine MH, as well as its relation to other disorders in the dog, and to MH in other species.
To resolve these issues, Thomas E. Nelson assembled a pedigree for in vivo halothane/succinylcholine challenge and in vitro contracture testing (IVCT), beginning with a Doberman-German Shepherd-Collie mixed breed progenitor from a colony maintained by the late Dr. Barry Reynolds at the University of Saskatchewan.19 Members of the pedigree produced by the mating of this dog to an MHN (MH Normal) Labrador Retriever dam were challenged under well-controlled anesthetic dosing regimens. Contemporary monitors and serial laboratory assays were used to discriminate features shared with human and swine MH from those that were distinct, and to establish the predictive value of the IVCT in a third species. Subsequently, a male subject in these experiments became the propositus of a second colony bred for molecular genetic diagnosis. His mates and all viable descendants were IVCT phenotyped to ascertain MH status, and in vivo halothane/succinylcholine challenge tests were simultaneously conducted in all dogs from 4 of the 6 sibships. Because swine and many human probands are predisposed to MHS by mutations in the calcium release channel gene (RYR1),20 co-segregation was sought between inheritance of the canine MHS trait and RYR1 as a candidate locus. Initial experiments ruled out the RYR1 mutation shared between all MHS swine (R614C) and 2-7% of human MHS (R615C) pedigrees.21
In one embodiment, the present invention is a method of determining whether a canine is susceptible to canine malignant hyperthermia. This invention is desirable because one would wish to test for this potentially life-threatening condition prior to surgery on a pet or valuable breeding or research animal.
The method preferably comprises the step of obtaining a nucleic acid sample from a canine and examining the sample for the presence or absence of a T1640C mutation, wherein the presence of the mutation indicates that the canine is susceptible to canine malignant hyperthermia. Preferably, the nucleic acid sample is a genomic DNA sample.
In another embodiment, the present invention is the method as described above wherein the step of examining the sample for the presence or absence of a T1640C mutation comprises amplifying a portion of the canine RYR1 locus and examining the amplified product for the presence of the mutation. Preferably, the method further comprises the step of digesting the amplification product with a restriction endonuclease, most preferably Mscl.
It is an object of the the present invention to provide a method of determining whether a canine is susceptible to canine malignant hyperthermia.
Other objects, features and advantages of the present invention will become apparent after one has reviewed the specifications, claims and drawings.